Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor involved in various cellular biochemical reactions and contributes to the regulation of Ca2+ signaling, chromatin structure, DNA repair and lifespan. To date, the signaling pathways that regulate NAD+ homeostasis remain unclear due to the dynamic nature and complexity of the NAD+ metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that plays important roles in the maintenance of NAD+ pool as well as calorie restriction (CR)-induced lifespan. In this proposal, we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD+ metabolism. Our preliminary results show that the phosphate responsive signaling (PHO) pathway contributes to the control of NAD+ metabolism. We have also identified additional novel components in the NmR/NAD+ biosynthesis and homeostasis pathways. The current proposal builds on our recent studies of these factors and the interplay between components in NmR/NAD+ metabolism and the nutrient signaling pathways. The long-term goal of our research is to understand the mechanisms by which yeast and mammalian cells maintain NAD+ homeostasis in response to changes in growth conditions. The major hypothesis is that NAD+ homeostasis is modulated by nutrient-sensing signaling pathway(s), which plays an important role in determining cell fitness and survival. The specific aims of the projects are: Aim 1) To study the role of nutrient sensing pathways in NmR and NAD+ homeostasis, Aim 2) To characterize a putative NmR assimilating enzyme and to study its role in NAD+ metabolism and CR, Aim 3) To study the roles of a conserved NmR homeostasis factor Fun26 in NAD+ metabolism and CR, and Aim 4) To study the role of the human Fun26 orthologs in NAD+ homeostasis and CR. To achieve these goals we will employ a combination of molecular, genetic and biochemical methods to analyze genes, proteins and pathways involved. These studies will increase our understanding of how eukaryotic cells regulate NAD+ homeostasis in response to changes in growth conditions, and which of the nutrient sensing signaling pathways are involved. Our findings may also contribute to the understanding of the molecular basis of the regulation of NAD+ homeostasis as well as metabolic disorders related to aberrant NAD+ metabolism in human. PUBLIC HEALTH RELEVANCE: Niacin (vitamin B3, a NAD+ precursor) is widely used as a food supplement to improve heath. Administration of NAD+ precursors has also been shown to ameliorate human deficiencies related to aberrant NAD+ metabolism. This research utilizes the genetically amenable model system, budding yeast, and mammalian cells to study the regulation of NAD+ homeostasis and signaling. Many components in NAD+ biosynthesis and metabolic pathways are conserved from yeast to mammals. Therefore, our findings are likely to provide insight into the development of therapeutic reagents for NAD+ deficiency-associated diseases such as diabetes and cancers, and also have broad implications for improving human nutrition and health.